Amphotropic retrovirus packaging cell line, process for its production and use thereof

ABSTRACT

A titer of retroviral vectors of at least about 107 colony-forming units/ml cell culture supernatants is obtained using an amphotropic retroviral packaging cell line which contains functional gag, pol and env genes integrated into the genome in which the expression of the genes gag and pol is regulated independently of the expression of the env genes, wherein the genome of the said cell line contains one or two functionally active env genes.

The invention concerns an improved amphotropic retroviral packaging cellline, a process for its production as well as its use especially forgene therapy in vitro and in vivo. High virus titres can be achievedwith retroviral vectors which are produced by such a packaging cellline.

Retroviral vectors are widely used to transfer foreign genes into cellsof mammals and humans. However, for safety reasons such retroviralvectors should no longer be replication-competent. For this reasonpackaging cell lines are used to produce retroviral vectors which arenot able to produce wild-type virus since they contain no functionalpackaging sequences. Such packaging cell lines have been known anddescribed for a long time: Ψ2 (Mann et al., Cell 33 (1983) 153-156),Ψ-Am (Cone and Mulligan, Proc. Natl. Acad. Sci. USA 81 (1994)6349-6353), PA12 (Miller et al., Mol. Cell Biol. 5 (1985) 431-437),GP+E-86 (Markowitz et al., J. Virol. 62 (1988) 1120-1124), PA317 (Millerand Buttimore, Mol. Cell Biol.6 (1986) 2895-2902), and GP+env Am12(Markowitz et al., Virology 167 (1988) 400-406), see also “Handbuch dermolekularen Medizin”, Vol. 1 Ed. D. Ganten and K. Ruckpaul, SpringerPublishers, Heidelberg 1997, R. Rüger, chapter 2.1, 197-241.

However, all these packaging cell lines except for GP+env Am12 (referredto as Am12 in the following) lead to a certain extent to the formationof wild-type virus (Bosselman et al., Mol.Cell Biol. 7 (1987)1797-1804). In contrast Am12 in which the viral gag and pol functionsare introduced into the cell on one plasmid and env is introduced intothe cell on another plasmid, is a safe packaging cell line. Thesefunctions are therefore integrated in different loci of the packagingcell genome. In order to form Nvld-type virus from Am12 threerecombinations have to occur which restore the original intact genome.This appears to be almost impossible and also has previously not beenobserved.

In order to avoid the problems caused by homologous recombination inpackaging cell lines attempts have for example also been made to expressgag/pol and env by different promoters (Meyers et al., Arch.Virol. 119(1991) 257-264; Dougherty et al., J.Virol. 63 (1989) 3209-3212) and touse packaging cell lines based on spleen necrosis virus (SNV) (Mlartinezand Dornburg, Virology 208 (1995) 234-241).

However, a disadvantage of the known packaging cell lines is that thevector-virus titres of the retroviral vectors produced in this mannerare very low and range between 10² and a maximum of 10⁶ colony-formingunits (CFU)/ml cell culture supernatant.

The object of the invention is to provide retroviral, safe packagingcell lines which are able to produce vector viruses with a hightransduction efficiency.

The invention concerns an amphotropic retroviral packaging cell linewhich contains functional gag, pol and env genes integrated into thegenome in which the expression of the genes gag and pol is regulatedindependently of the expression of the env genes characterized in thatthe genome of the said cell line contains one or two functionally activeenv genes.

A further subject matter of the invention is a process for producing aretroviral vector characterized in that an amphotropic retroviralpackaging cell line according to the invention is transfected with avector-virus (vector genome) which contains one or more genes that areheterologous for the virus but contains no functionally activeretroviral structural genes in which the cell line is cultured and theretroviral vector is isolated from the culture supernatant.

A further subject matter of the invention is a process for producing anamphotropic retroviral packaging cell line characterized in that an envhelper plasmid containing a selection gene and a gag/pol helper plasmidcontaining a selection gene are transfected into a eukaryotic cell,transfected cells are identified on the basis of the selection gene andare isolated and those cells are selected which contain one or twofunctionally-active env genes.

A further subject matter of the invention is a replication-incompetentretroviral vector virus which has been produced by the method accordingto the invention.

It has turned out that the number of env integrates in the genome of apackaging cell line has a decisive influence on the transductionefficiency of the vector-viruses produced in this manner. This is allthe more surprising since it is known from Martinez and Dornburg,Virology 208 (1995) 234-241 that the extent of env expression in apackaging cell line would not have an influence on the transductionefficiency. The packaging cell lines examined by Martinez and Dornburgdiffer with respect to env expression only in the promoter strength ofthe sequences regulating env expression.

In a preferred embodiment the packaging cell line according to theinvention contains two env integrates and a gag/pol integrate. Accordingto the invention use of the packaging cell line achieves a titre ofretroviral vectors of 2 10⁶ to 10⁷ or more, preferably at least 10⁷colony-forming units/ml cell culture supernatant and/or preferably atransduction efficiency of at least 50% in MOLT-4 cells (ATCC CRL 1582)after three days measured by the centrifugation method.

The independent regulation of the gene expression of gag/pol and env isusually achieved by locating at least one expression control sequenceand/or a stop signal between gag/pol and env.

Starting cell lines for the packaging cell line are for example 3T3,D17. Suitable starting vectors are based on, for example, MLV, MoMuLV.Such suitable packaging cell lines and starting vectors are known to aperson skilled in the art and are described for example by R. Rüger(1997) in “Handbuch der molekularen Medizin”.

The cell line HSR BM01 is particularly preferred as a packaging cellline according to the invention. This cell line was deposited on Dec. 9,1995 by Boehringer Mannheim GmbH according to the Budapest Contract inthe “Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH” (DSM),Mascheroder Weg 1b, 38124 Braunschweig under the No. DSM ACC 2235.

A further subject matter of the invention is a process for producing anamphotropic retroviral packaging cell line according to the invention.

An amphotropic split genome retroviral packaging cell line can forexample be produced as described in Markowitz et al., Virology 167(1988) 400-406. Two plasmids are used for this which code for theso-called f helper sequences (gag, pol and env).

In order to select cell lines according to the invention, the number ofenv and gag/pol integrates has to be determined. This is expedientlycarried out by digesting the genomic DNA with restriction enzymes whichdo not cleave in the gag/pol sequences or env sequences and determiningthe number of fragments which contain gag/pol- or env-specificsequences.

The invention whose protective scope results from the patent claims isfurther elucidated by the following examples and publications. Thedescribed methods are to be understood as examples which still describethe subject matter of the invention even after modifications.

EXAMPLE 1

Construction of Helper Plasmid

Construction of the gag/pol Helper Plasmid

A helper plasmid carries the viral structural genes gag/pol of theMoloney murine leukaemia virus (MoMULV) e.g. under the control of theMoMULV 5′LTR. These structural genes gag/pol together with the 5′LTR canbe obtained from the proviral DNA of MoMULV by restriction digestion. Inthis process it is important for safety reasons to inactivate theso-called packaging sequences Ψ. This can for example be achieved by adeletion which is introduced with the aid of a restriction digestion.Furthermore it is advantageous to have a selectable marker such as e.g.the gpt gene on the helper plasmid the gene product of which impartsresistance to the selection drug mycophenolic acid. Mycophenolic acidresistance allows the rapid selection of clones which have taken up thedesired plasmid (gag/pol helper plasmid). A corresponding helper plasmidcan be constructed as described for example by Markowitz et al., J.Virol. 62 (1988) 1120-1124.

Construction of the env Helper Plasmid

Another helper plasmid carries the viral structural gene env of theamphotropic murine leukaemia virus clone 4070A (4070A) (Chattopadhyay etal., J. Virol. 39 (1981) 777-791) e.g. under the control of MoMULV5′LTR. For this a fragment of the proviral DNA of 4070A which containsthe env gene and the 3′ acceptor splice site is isolated. This fragmentis cloned into a plasmid which contains the MoMULV 5′LTR as well as the5′ donor splice site. A corresponding helper plasmid can be produced asdescribed for example by Markowitz et al., Virology 167 (1988) 400-406.

EXAMPLE 2

Transfer of the Helper Constructs

Transfer of the gag/pol Helper Plasmid

NIH 3T3 cells (ATCC No. CRL 1658) are for example firstly transfectedwith the gag/pol helper plasmid. This can for example be carried outwith the aid of electroporation. Transfected cells can be isolated withthe aid of a suitable selection medium since the gpt gene is present onthe gag/pol plasmid. A suitable selection medium contains for examplehypoxanthine (15 μg/ml), xanthine (250 μg/ml) and mycophenolic acid (25μg/ml) (HXM medium). Resistant clones can be subsequently examined forthe expression of reverse transcriptase (RT) as for example described byMarkowitz et al., J.Virol. 62 (1988) 1120-1124. A clone that expressesto a particularly high degree is selected for subsequent transfectionwith the env helper plasmid.

Transfer of the env Helper Plasmid

Subsequently the env helper plasmid can be transfected for example withthe aid of electroporation into a gag/pol-transfected, mycophenolicacid-resistant clone with high RT activity. In order to simply identifysuccessfully transfected cells it is advantageous to for exampleco-transfect a further plasmid which contains a resistant gene togetherwith the helper plasmid. This can for example be a plasmid which impartsresistance to hygromycin such as for example the plasmid pRSHhyg(Murphy, A. J. (1987) doctoral thesis, Columbia University, USA).Successfully transfected clones can be simply selected with the aid of asuitable selection medium which for example contains 200 μg/mlhygromycin B. Hygromycin-resistant clones can be subsequently examinedfor env expression with the aid of radioimmuno-precipitation using anenv antiserum as for example described in Markowitz et al., Virology 167(1988) 400-406.

EXAMPLE 3

Selection of Amphotropic Retroviral Packaging Cell Lines

The number of copies of the env helper plasmid integrated into thegenome was selected as a parameter for choosing a certain subclone of apackaging cell line. Martinez and Dornburg, Virology 208 (1995) 234-241have showed that, in contrast to gag/pol, a higher env expression has noinfluence on the virus titre; the titre of clones with a higher gag/polbut not a higher env expression correlated with the found infectionefficiency. This was the basis for the assumption that the ratio ofgag/pol to env gene products may be very important for the efficiency ofpackaging cells and possibly packaging cells with a lower env expressionmay lead to viral supernatants with a higher transduction efficiency.

Determination of the Number of env Integrates

The number of copies of the env helper plasmid integrated into thegenome of the individually examined clones was determined with the aidof restriction digestion and Southern blot analysis. For this genomicDNA was isolated from various subclones and digested with the aid ofrestriction enzymes e.g. the enzyme PstI. This enzyme does not cleave inthe env sequence and only cleaves once in the entire plasmid sequence.Since each corresponding second cleavage site which leads to therespective restriction fragment is located randomly in the genome, it ispossible in this manner to determine the number of env integrates.

The digested DNA was separated in an Agarose gel and transferred onto anylon membrane by means of capillary transfer. The fragments whichcontain env-specific sequences were detected with the aid of anenv-specific, digoxigenin-labelled DNA probe. Subclones with differentnumbers of env integrates were found. For example the band sizes thatwere obtained after digestion with the restriction enzyme PstI were 17.5kb, 13.3 kb and 3.4 kb for the packaging cell line GP+env Am12 and 13.3kb and 3.4 kb for the packaging cell line HSR BM01.

EXAMPLE 4

Production of Virus-producing Cell Lines with Different Numbers of envIntegrates

Each subclone was transfected either with two (HSR BM01) or three(GP+env Am12) env integrates containing the retroviral construct pL1 inorder to produce virus-producing cell lines (producer lines). Theconstruct pL1 carries the cDNA for a shortened form of the human lowaffinity nerve growth factor receptor (hΔLNGFR (produced analogously toWO 95/06723)) in which the cytoplasmic domain is deleted and which isunder the control of the viral 5′=0 LTR. The gene product of the hΔLNGFRcDNA, a membrane protein, can be detected on the cells with the aid of ahΔLNGFR-specific monoclonal antibody (mAb). Virus-producing clones wereisolated from HSR BM01 (HSRBM-L1) as well as from GP+env Am12 (cloneC11-C4) with the aid of immunofluorescence and flow cytometry.

Determination of the Titre of Supernatants from Virus-Producing CellsContaining Different Numbers of env Integrates

Indicator cells (NIH/3T3; ATCC No. CRL 1658) were transduced with thesupernatants containing virus particles from the two clones HSRBM-L1 orC11-C4. A cytochemical staining method based on the anti-hΔLNGFR mAballows the determination of the number of colony-forming units (titre)of hΔLNGFR-coding retroviruses. Whereas the supernatant of the producerline C11-C4 (3 env copies) based on the packaging cell GP+env Am12 had atitre of 6.3×10⁵ colony-forming units, the supernatant of the producerline HSRBM-L1 (two env copies) based on HSR BM01 had a titre of1.25×10⁷.

EXAMPLE 5

Transduction of Various Cell Lines with Supernatants from Two DifferentProducer Cell Lines which Contain the Same Retroviral Vector

The cell lines Jurkat, MOLT4, Raji and K 562 are cultured in RPMI/10%FCS under normal cell culture conditions and transduced at celldensities of 3-9×10⁵ cells/ml. The various producer cell lines whichcontain an identical retroviral vector are cultured in DMEM/10% FCSunder normal cell culture conditions. The cells are seeded at 1×10⁴cells/cm². After three days the supernatant is discarded and freshmedium is added. The supernatant is harvested 24 hours later. Thesupernatant is filtered through a 0.22 μm filter. The titre of thevarious supernatants is determined and adjusted such that equivalentvolume amounts contain an equivalent number of virus particles. Thetitre of the retroviral supernatant was determined using the method ofimmune staining titration using NIH3T3 as target cells.

Transduction

Protocol 1

5×10⁵ cells are resuspended in 1 ml retroviral supernatant. Thetransduction is carried out in a 24-well plate. After centrifugation(1000 g, 30° C., 90 min.) the cells are washed and resuspended in 1 mlfresh cell culture medium. The cells are incubated overnight and 1 mlfresh medium is added. Three days after transduction the cells areharvested and stained with an FITC-labelled anti-LNGFR antibody. Thepercentage of LNGFR-expressing cells is determined by means of flowcytometry. In this process dead cells are excluded from the analysis bypropidium iodide staining. The flow-cytometry analysis is carried out ona FACScan instrument.

Protocol 2

The procedure in protocol 2 was carried out according to protocol 1, butwithout centrifugation. The cells are incubated for two hours at 37° C.with the retroviral supernatant.

Table 1 shows the LNGFR expression of various cell lines three daysafter transduction according to protocol 1.

Table 2 shows the LNGFR expression of various cell lines three daysafter transduction according to protocol 2.

TABLE 1 % LNGFR-positive cells three days after transduction(centrifugation method) HSR BM01 GP + envAm12 (HSRBM-L1) (clone C11-C4)K562 88.81 12.79 MOLT4 53.64 3.52 RAJI 25.31 8.83 JURKAT 44.36 3.96

TABLE 2 % LNGFR-positive cells three days after transduction (incubationmethod) HSR BM01 GP + envAm12 (HSRBM-L1) (clone C11-C4) K562 68.69 8.24MOLT4 37.86 2.68 RAJI 6.72 3.01 JURKAT 23.91 4.64

LIST OF REFERENCES

Bosselman et al., Mol. Cell Biol. 7 (1987) 1797-1804

Chattopadhyay et al., J. Virol. 39 (1981) 777-791

Cone and Mulligan, Proc. Natl. Acad. Sci. USA 81 (1994) 6349-6353

Dougherty et al., J. Virol. 63 (1989) 3209-3212

Handbuch der molekularen Medizin, Volume I, Ed. D. Ganten and K.Ruckpaul, Springer Verlag Heidelberg 1997, R. Rüger, Chapter 2.1,197-241

Mann et al., Cell 33 (1983) 153-156

Markowitz et al., J. Virol. 62 (1988) 1120-1124

Markowitz et al., Virology 167 (1988) 400-406

Martinez and Dornburg, Virology 208 (1995) 234-241

Meyers et al., Arch. Virol. 119 (1991) 257-264

Miller and Buttimore, Mol. Cell Biol. 6 (1986) 2895-2902

Miller et al., Mol. Cell Biol. 5 (1985) 431-437

Murphy, A. J. (1987), Doctoral Thesis, Columbia University, USA

What is claimed is:
 1. An amphotropic retroviral packaging cell line,each packaging cell containing exactly one functional gag gene, exactlyone functional pol gene and exactly two functional env genes, the genesbeing integrated into the genome in such a manner that the expression ofthe gag and pol genes is regulated independently of the expression ofthe env genes and a titre of at least 10⁷ colony-forming units/ml cellculture supernatant is achieved by the packaging cell line whenpackaging a retroviral vector.
 2. A process for producing an amphotropicretroviral packaging cell line, which comprises transfecting aeukaryotic cell with an env helper plasmid containing a selection geneand a gag/pol helper plasmid containing a selection gene, identifyingthe transfected cells on the basis of the selection gene, and isolatingthose cells which contain two functionally active env genes.
 3. Thepackaging cell line DSM ACC
 2235. 4. A process for the production of aretroviral vector, which comprises (i) transfecting a eukaryotic cellwith an env helper plasmid containing a selection gene and a gag/polhelper plasmid containing a selection, (ii) identifying the transfectedcells on the basis of the selection gene, (iii) isolating those cellswhich contain two functionally active env genes, (iv) transfecting theisolated cells with a vector genome which contains one or moreheterologous genes for the virus but contains no functionally activeretroviral structural genes, (v) culturing the cells, and (vi) isolatingthe retroviral vector from the culture supernatant.